APPENDIX II-AH: WNV and Blood Brain Barrier Damage Caused by Pesticides-CCHE Research of Peer-Reviewed Articles: Ben-Nathan, et al, CNS penetration by noninvasive viruses following inhalational anesthetics., Ann N Y Acad Sci 2000;917:944-50; Ben-Nathan, et al, Cold stress-induced neuroinvasiveness of attenuated arboviruses is not solely mediated by Corticosterone, Arch Virol 1996;141(7):1221-9; 1; Kolesnichenko,et al, [Changes in the biochemical composition of the cerebrospinal fluid in acute carbophos poisoning], Zh Nevropatol Psikhiatr Im S S Korsakova 1992;92(2):95-9; Pavlovsky, et al, Pyridostigmine enhances glutamatergic transmission in hippocampal CA1 neurons, Exp Neurol 2003 Feb;179(2):181-7; Lallement , et al, Review of the value of huperzine as pretreatment of organophosphate poisoning, ; Neurotoxicology 2002 May;23(1):1-5 ; Pall, et al, Elevated nitric oxide/peroxynitrite mechanism for the common etiology of multiple chemical sensitivity,chronic fatigue syndrome, and posttraumatic stress disorder, Ann N Y Acad Sci 2001 Mar;933:323-9 ; Cowan , et al, Gene therapy to prevent organophosphate intoxication, Toxicol Appl Pharmacol 2001 May 15;173(1):1-6; Gupta, et al, Functional impairment of blood-brain barrier following pesticide exposure during early development in Rats, Hum Exp Toxicol 1999 Mar;18(3):174-9; Friedman , et al, Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response, , Nat Med 1996 Dec;2(12):1382-5 , Comment in: * Nat Med. 1996 Dec;2(12):1307-8. * Nat Med. 1997 Apr;3(4):366. * Nat Med. 1997 Mar;3(3):253; Carpentier, et al, Seizure-related opening of the blood-brain barrier induced by soman: possible correlation with the acute neuropathology observed in poisoned rats, Neurotoxicology 1990 Fall;11(3):493-508.

 

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Blood brain barrier and pesticides

1

: Ann N Y Acad Sci 2000;917:944-50 Related Articles, Links

CNS penetration by noninvasive viruses following inhalational anesthetics.

[Note this one shows the brain invasion of non-invasive WNV because anaesethetic or 30% CO2 cause stress,

breaching BBB and allowing WNV to cause encephalitis.]

Ben-Nathan D, Kobiler D, Rzotkiewicz S, Lustig S, Katz Y.

Dept. of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel. ben@iibr.gov.il

The effects of inhalational anesthetics on brain penetration by the neurovirulent noninvasive West Nile virus (WN-

25) were studied in mice. WN-25 injected intracerebrally causes encephalitis and kills adult mice, but when injected

intraperitoneally (i.p.) it is unable to invade the brain and kill. Under stress conditions, this strain causes encephalitis

and death even after i.p. inoculation. In the study described in this paper, we used two inhalational anesthetics, a

single short-term exposure to 2% halothane for 10 min in oxygen, or 70% nitrous oxide (N2O) for 30 min in air. Both

inhalational anesthetics induced WN-25 encephalitis and death in 33% and 20% of the tested mice, respectively.

Exposure of inoculated mice to halothane for prolonged periods or for repeated exposures (two or three times)

markedly increased the mortality rate (up to 75%). Exposure to 30% CO2, a known modulator of blood-brain barrier

(BBB) activity, was used as a positive control (80% mortality). No death was observed in the control non-exposed

injected mice. Virus levels were found to be more than 10(7) plaque-forming units (PFU)/brain in all moribund mice.

Additional parameter demonstrating the "stressor-like" nature of inhalation anesthetics was the induction of a

significant decrease in weight of the lymphoid organs of inoculated mice. We suggest that inhalational anesthetics

induces BBB breaching with subsequent entrance of the noninvasive WN-25 virus into the brain, causing encephalitis

and death.

PMID: 11268425 [PubMed - indexed for MEDLINE]

Arch Virol 1996;141(7):1221-9 Related Articles, Links

Cold stress-induced neuroinvasiveness of attenuated arboviruses is not solely mediated by

corticosterone.

[Note this one by the same researchers shows that corticosteroids do not provide sufficient stress on the brain

blood barrier to allow the non-invasive WNV to pass]

Ben-Nathan D, Lustig S, Kobiler D.

Department of Virology, Israel Institute for Biological Research, Ness-Ziona, Israel.

In previous studies we have shown that various stress paradigms can induce the penetration of noninvasive,

attenuated viruses into the central nervous system (CNS). Since glucocorticoids levels are elevated during stress, we

compared the effect of cold stress and corticosterone (CS) injection on neuroinvasiveness of a non-invasive

encephalitic virus, WN-25 (West Nile). Exposure of inoculated mice to cold stress or CS resulted in high viremia and

a marked increase in mortality when compared to control untreated mice. Exposure of WN-25 inoculated mice to cold

treatment or CS injection led to high blood virus levels as compared to nontreated mice (3.2 and 3.1 vs > 1 log 10

PFU/ml). Cold stress or CS (5000 ng/mouse) treatment caused a mortality rate of 70% and 50% of the WN-25

inoculated mice respectively. No mortality was recorded in control inoculated groups (p < 0.05). Passive transfer

serum from uninfected cold stressed mice to WN-25 inoculated nonstressed mice, resulted in similar mortality. The

levels of CS in passive transferred serum from cold stressed animals was 500 ng/ml, only 2% (100 vs. 5000 ng) of

the CS dose required to obtain a similar effect on viral penetration and mortality when CS was injected directly.

Therefore, we concluded that CS was not the sole factor responsible for the cold stress effect on the viral infection

outcome.

PMID: 8774683 [PubMed - indexed for MEDLINE]

2

1: Zh Nevropatol Psikhiatr Im S S Korsakova 1992;92(2):95-9 Related Articles, Links

[Changes in the biochemical composition of the cerebrospinal fluid in acute carbophos poisoning]

[Article in Russian]

Kolesnichenko IP, Dolgo-Saburova IS, Somova TV.

As far as the pathogenesis of poisonings with organophosphorus pesticides is concerned, in addition to irreversible

inhibition of acetylcholinesterase (AGE) in tissues, of importance are changes in the other systems which essentially

determine the outcome of intoxication. The purpose of the present study was to examine the nature of changes

occurring in total protein and protein fractions, free amino acids (aspartic and glutamic acids, glycine, isoleucine,

leucine) and in certain enzymes (AST, ALT, CP, GGTP, GDH) in the cerebrospinal fluid (CSF) of patients with acute

Malathion insecticide poisoning. 137 patients aged 20 to 50 years were placed under observation. There were 77

men and 60 women. 40 persons had poisoning of medium gravity and 97 were severely poisoned. The intake of the

CSF was performed on days 1, 3, 10, 14 and 21 since the disease onset. It has been established that in acute

Malathion insecticide poisoning, the CSF content of the stimulating mediator amino acids, aspartic and glutamic, rises

within the early periods, whereas the concentration of the inhibitory mediator glycine decreases. The changes in

protein fractions of the CSF are characterized by a fall of the content of globulins and a rise of albumins, thus

attesting to the predominance of pathological processes in the brain, especially in the initial period of intoxication, and

to the impairment of the blood-brain barrier. The development of intoxication is associated with activation in the CSF

of LDN, CP, GGTP and GDH as well as by activation of LDH isozymes which is viewed as the result of the

membranotoxic effect of a Malathion insecticide.

PMID: 1355942 [PubMed - indexed for MEDLINE]

Exp Neurol 2003 Feb;179(2):181-7 Related Articles, Links

Pyridostigmine enhances glutamatergic transmission in hippocampal CA1 neurons.

Pavlovsky L, Browne RO, Friedman A.

Department of Physiology, Soroka University Medical Center, Ben-Gurion University and Zlotowski Center of

Neuroscience, Beersheva, Israel.

Pyridostigmine, a carbamate acetylcholinesterase (AChE) inhibitor, is routinely employed in the treatment of the

autoimmune disease myasthenia gravis. Due to its positively charged ammonium group, under normal conditions

pyridostigmine cannot cross the blood-brain barrier (BBB) and penetrate the brain. However, several studies have

suggested that under conditions in which the BBB is disrupted, pyridostigmine enters the brain, changes cortical

excitability, and leads to long-lasting alterations in gene expression. The aim of this study was to characterize the

mechanisms underlying pyridostigmine-induced changes in the excitability of central neurons. Using whole cell

intracellular recordings in hippocampal neurons we show that pyridostigmine decreases repetitive firing adaptation

and increases the appearance of excitatory postsynaptic potentials. In voltage clamp recordings, both pyridostigmine

and acetylcholine (ACh) increased the frequency but not the amplitude of excitatory postsynaptic currents. These

effects were reversible upon the administration of the muscarinic receptor antagonist, atropine, and were not blocked

by tetrodotoxin. We conclude that pyridostigmine, by increasing free ACh levels, causes muscarinic-dependent

enhancement of excitatory transmission. This mechanism may explain central side effects previously attributed to this

drug as well as the potency of AChE inhibitors, including nerve-gas agents and organophosphate pesticides, in the

initiation of cortical synchronization, epileptic discharge, and excitotoxic damage.

PMID: 12618125 [PubMed - indexed for MEDLINE]

Blood brain barrier and pesticides

3

Neurotoxicology 2002 May;23(1):1-5 Related Articles, Links

Review of the value of huperzine as pretreatment of organophosphate poisoning.

Lallement G, Baille V, Baubichon D, Carpentier P, Collombet JM, Filliat P, Foquin A, Four E, Masqueliez C,

Testylier G, Tonduli L, Dorandeu F.

Unite de Neuropharmacologie, CRSSA, La Tronche, France. guylallement@compuserve.com

Today, organophosphate (OP) nerve agents are still considered as potential threats in both military or terrorism

situations. OP agents are potent irreversible inhibitors of central and peripheral acetylcholinesterases. Pretreatment

of OP poisoning relies on the subchronic administration of the reversible acetylcholinesterase (AChE) inhibitor

pyridostigmine (PYR). Since PYR does not penetrate into the brain, it does not afford protection against seizures and

subsequent neuropathology induced by an OP agent such as soman. Comparatively, huperzine (HUP) is a reversible

AChE inhibitor that crosses the blood-brain barrier. HUP is presently approved for human use or is in course of

clinical trials for the treatment of Alzheimer's disease or myasthenia gravis. HUP is also used as supplementary drug

in the USA for correction of memory impairment. Besides, HUP has also been successfully tested for pretreatment of

OP poisoning. This review summarizes the therapeutical value of HUP in this field. Moreover, the modes of action of

HUP underlying its efficacy against OP agents are described. Efficacy appears mainly related to both the selectivity

of HUP for red cell AChE which preserves scavenger capacity of plasma butyrylcholinesterases for OP agents and to

the protection conferred by HUP on cerebral AChE. Finally, recent data, showing that HUP seems to be devoid of

deleterious effects in healthy subjects, are also presented. Globally, this review reinforces the therapeutical value of

HUP for the optimal pretreatment of OP poisoning.

Publication Types:

* Review

* Review, Tutorial

PMID: 12164543 [PubMed - indexed for MEDLINE]

Ann N Y Acad Sci 2001 Mar;933:323-9 Related Articles, Links

Elevated nitric oxide/peroxynitrite mechanism for the common etiology of multiple chemical sensitivity,

chronic fatigue syndrome, and posttraumatic stress disorder.

Pall ML, Satterle JD.

School of Molecular Biosciences, Washington State University, Pullman 99164-4660, USA. martin_pall@wsu.edu

Various types of evidence implicate nitric oxide and an oxidant, possibly peroxynitrite, in MCS and chemical

intolerance (CI). The positive feedback loops proposed earlier for CFS may explain the chronic nature of MCS (CI) as

well as several of its other reported properties. These observations raise the possibility that this proposed elevated

nitric oxide/peroxynitrite mechanism may be the mechanism of a new disease paradigm, answering the question

raised by Miller earlier: "Are we on the threshold of a new theory of disease?"

Publication Types:

* Review

* Review, Tutorial

PMID: 12000033 [PubMed - indexed for MEDLINE]

4

Toxicol Appl Pharmacol 2001 May 15;173(1):1-6 Related Articles, Links

Gene therapy to prevent organophosphate intoxication.

Cowan J, Sinton CM, Varley AW, Wians FH, Haley RW, Munford RS.

Molecular Host Defense Laboratory, Division of Infectious Diseases, Department of Internal Medicine, University of

Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

The specific hydrolytic activity of PON1 paraoxonase/arylesterase enzymes in liver and blood provides a natural

barrier against the entry of organophosphate toxins into the central and peripheral nervous systems. Inherited

differences in PON1 enzyme concentrations may determine levels of susceptibility to organophosphate injury in

humans. To test whether boosting serum levels of PON1 enzymes by gene therapy might provide increased

protection, we compared the degree of inactivation of whole brain acetylcholinesterase of mice exposed to

chlorpyrifos 4 days after intravenous injection of recombinant adenoviruses containing PON1-LQ or PON1-LR genes

or no PON1 gene. Both recombinant viruses containing PON1 genes boosted serum arylesterase concentrations by

approximately 60% and significantly prevented the inactivation of brain acetylcholinesterase. Some mice were

completely protected. These findings indicate that boosting serum levels of PON1 enzymes by a gene delivery vector

raises the threshold for organophosphate toxicity by hydrolytic destruction before the chemical can enter the brain.

Copyright 2001 Academic Press.

PMID: 11350209 [PubMed - indexed for MEDLINE]

Hum Exp Toxicol 1999 Mar;18(3):174-9 Related Articles, Links

Functional impairment of blood-brain barrier following pesticide exposure during early development in

rats.

Gupta A, Agarwal R, Shukla GS.

Predictive Toxicology Research Group, Industrial Toxicology Research Centre, Lucknow, India.

1. The effect of certain pesticides on the functional integrity of the developing blood-brain barrier (BBB) was

studied following single and repeated exposure, and after subsequent withdrawal in rats. 2. Ten-day-old rat pups

exposed orally to quinalphos (QP, organophosphate), cypermethrin (CM, pyrethroid) and lindane (LD,

organochlorine) at a dose of 1/50th of LD50, showed a significant increase in the brain uptake index (BUI) for a

micromolecular tracer, sodium fluorescein (SF), by 97, 37 and 72%, respectively, after 2 h. Residual increases in the

BUI were found even after 3 days of the single treatment of QP (28%) and LD (23%). 3. Repeated exposure for 8

days (postnatal days (PND) 10-17) with QP, CM and LD increased the BBB permeability by 130, 80 and 50%,

respectively. Recovery from these changes was complete in QP and LD-treated animals after 13 days (PND 18-30)

of withdrawal. However, CM showed persistent effects that were normalized only after 43 days (PND 18-60) of

withdrawal. 4. A single dose reduced to 1/100th of LD50 also increased BUI in 10-day-old rat pups following QP

(20%) and CM (28%) exposure at 2 h. 5. An age-dependent effect of these pesticides was evident from the study

showing higher magnitude of BUI changes in 10-day-old rats as compared to that in 15-day-old rats. Furthermore,

adult rats did not show any effect on BBB permeability even at a higher dose (1/25th of LD50) of these pesticides

given alone or in combination with piperonyl butoxide (600 mg/kg, i.p.) for 3 consecutive days. 6. This study showed

that developing BBB is highly vulnerable to single or repeated exposure of certain pesticides. The observed

persistent effects during brain development even after withdrawal of the treatment may produce some neurological

dysfunction at later life as well.

PMID: 10215108 [PubMed - indexed for MEDLINE]

Blood brain barrier and pesticides

5

Nat Med 1996 Dec;2(12):1382-5 Related Articles, Links

Comment in: * Nat Med. 1996 Dec;2(12):1307-8. * Nat Med. 1997 Apr;3(4):366. * Nat Med. 1997 Mar;3(3):253.

Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate

transcriptional response.

Friedman A, Kaufer D, Shemer J, Hendler I, Soreq H, Tur-Kaspa I.

Department of Biological Chemistry, Life Sciences Institute, Hebrew University, Jerusalem, Israel.

Pyridostigmine, a carbamate acetylcholinesterase (AChE) inhibitor, is routinely employed in the treatment of the

autoimmune disease myasthenia gravis. Pyridostigmine is also recommended by most Western armies for use as

pretreatment under threat of chemical warfare, because of its protective effect against organophosphate poisoning.

Because of this drug's quaternary ammonium group, which prevents its penetration through the blood-brain barrier,

the symptoms associated with its routine use primarily reflect perturbations in peripheral nervous system functions.

Unexpectedly, under a similar regimen, pyridostigmine administration during the Persian Gulf War resulted in a

greater than threefold increase in the frequency of reported central nervous system symptoms. This increase was not

due to enhanced absorption (or decreased elimination) of the drug, because the inhibition efficacy of serum butyrylcholinesterase

was not modified. Because previous animal studies have shown stress-induced disruption of the

blood-brain barrier, an alternative possibility was that the stress situation associated with war allowed pyridostigmine

penetration into the brain. Here we report that after mice were subjected to a forced swim protocol (shown previously

to simulate stress), an increase in blood-brain barrier permeability reduced the pyridostigmine dose required to inhibit

mouse brain AChE activity by 50% to less than 1/100th of the usual dose. Under these conditions, peripherally

administered pyridostigmine increased the brain levels of c-fos oncogene and AChE mRNAs. Moreover, in vitro

exposure to pyridostigmine increased both electrical excitability and c-fos mRNA levels in brain slices, demonstrating

that the observed changes could be directly induced by pyridostigmine. These findings suggest that peripherally

acting drugs administered under stress may reach the brain and affect centrally controlled functions.

PMID: 8946841 [PubMed - indexed for MEDLINE]

Neurotoxicology 1990 Fall;11(3):493-508 Related Articles, Links

Seizure-related opening of the blood-brain barrier induced by soman: possible correlation with the acute

neuropathology observed in poisoned rats.

Carpentier P, Delamanche IS, Le Bert M, Blanchet G, Bouchaud C.

Centre de Recherches du Service de Sante des Armees, Unite de Neurotoxicologie, La Tronche, France.

In rats poisoned with soman, an irreversible organophosphate anticholinesterase, acute changes in blood-brain

barrier (BBB) permeability to proteins were investigated, using Evans Blue (EB)-labelled serum albumin and

plasmatic gamma-immunoglobulin G (IgG) as indicators. Confirming previously published data, soman produced a

conspicuous seizure-related and reversible BBB opening which was greatest after 30 to 60 min of paroxysmal

electroencephalographic (EEG) discharges when signs of cerebral hyperactivity (epileptic EEG pattern, hyperoxia)

were also at their height. Topographically, the protein leakage was bilateral and restricted to anatomically defined

brain structures, some of which being thereafter sites of parenchymal edema and neuronal damage. In these areas

(e.g., the thalamus), the edema is probably, at least in part, "vasogenic" in origin, and the possible contribution of the

transient BBB opening to the neuronal lesions was questioned. On the other hand, the hippocampus, a region

preferentially affected by the soman-induced acute neuropathology, was always free of any protein leakage,

suggesting that the edema is unrelated to vascular damage and "cytotoxic" in nature. Finally, no topographic

relationship was shown to exist between the increase in cerebrovascular permeability produced by soman and the

histochemically-detected inhibition of the parenchymal total cholinesterases (ChE) or endothelial

butyrylcholinesterase (BuChE).

PMID: 2284054 [PubMed - indexed for MEDLINE]